1. Field of the Invention
This invention relates to immunoassay methods for the quantitative determination of triiodothyronine (T.sub.3) and thyroxine (T.sub.4) in biological fluids. In particular, the present invention provides methods for the simultaneous measurement of these hormones by direct equilibrium dialysis and immunoassay.
2. Description of Related Art
The thyroid gland is an endocrine gland situated at the base of the neck which synthesizes two peptidic thyroid hormones, triiodothyronine (T.sub.3) and thyroxine (T.sub.4), by the condensation of two molecules of iodized thyrosine, monoiodothyrosine and diiodothyrosine. Thyroid hormone are synthesized and stored in the thyroid bound to the thyroid protein, thyroglobulin, until they are released into the circulation by a proteolytic process. The majority of thyroid hormone is then carried in the serum bound to three proteins: TBG (thyroxine-bonding globulin), TBPA (thyroxine-bonding prealbumin) and albumin. However, a small proportion (about 0.03% each of T.sub.3 and T.sub.4) of total thyroid hormone exists in the circulation in a free (unbound) form.
The circulating thyroid hormones exert their activity on several target tissues in the body. Thyroid hormones physiologically affect the tissues by stimulating cellular oxygen consumption, lipid and glucose metabolism, and influencing normal growth during development. However, it is generally assumed that it is only the free form, and not the bound form, of the thyroid hormones that can enter the cells to exert their physiological action.
A determination of free circulating thyroid hormone levels is critical to an appropriate assessment of thyroid function. The methods available for the quantitative determination of circulating concentrations of thyroid hormones are important for diagnosing hyperthyroidism or hypothyroidism. However, the determination of the total T.sub.4 or T.sub.3 levels as an indication of thyroid function may lead to erroneous diagnosis of thyroid disorder. For example, conditions which result in elevated levels of thyroid hormone binding proteins (such pregnancy, liver diseases and elevated estrogen or opiate levels) will proportionally result in elevated levels of bound thyroid hormones in the serum being detected in the assay. Elevated bound thyroid hormone levels may, thus, lead to a misdiagnosis of a patient as hyperthyroid, even where levels of free T.sub.3 or T.sub.4 in the serum may be normal. Therefore, it is important to be able to determine the level of free T.sub.3 and T.sub.4 which is biologically active in the patient to more accurately assess the status of the thyroid gland.
While considerable effort has been directed at the development of various assays for T.sub.3 and T.sub.4, there are a number of problems associated with existing assays for measuring these thyroid hormones. What is needed in the art is a simple assay which overcomes problems associated with existing assays and allows the simultaneous determination of free T.sub.3 and free T.sub.4 in a single serum sample. The invention described herein overcomes the problems associated with art accepted assays by providing simple and effective methods for the simultaneous immunological quantitative determination of the T.sub.3 and T.sub.4 thyroid hormones.